UNIT 1.13 Quantified Assessment of Terminal Density and Innervation This unit outlines a method for estimating the density of innervation and the size of the average terminal arbor of projection neurons. The basic premise is that the average arbor size (terminal tree, TT) can be obtained by dividing the total number of terminals (T) from projection neurons in the target nucleus by the number of projection neurons (N). In this unit, the dopaminergic (DA) innervation of the caudate putamen (CPu) from neurons of the substantia nigra pars compacta (SNpc) is used to illustrate the method. DA terminals in the CPu can be identified by their immunoreactivity for the dopamine transporter (DAT), and the projection neurons in the SNpc are identified by tyrosine hydroxylase (TH) immunoreactivity. Thus, counting the number of DAT-immunoreactive (DAT-ir) varicosities in the CPu provides a reasonable estimation of the number of terminals arising from the SNpc projection onto the CPu. UNIT 1.11 provides detailed instruction on the counting techniques that the protocols in this unit draw upon. This method was first developed using the most basic of equipment, that is, a projecting microscope with an oil lens. Currently the authors are using a Leica DML microscope fitted with a 100× 1.3 numerical aperture lens (PL Fluotar; Leica Microsystems) with Stereo Investigator (MicroBrightField) stereological systems. This system was chosen because at the time of purchase it provided the best resolution and its software was the easiest to use. The Stereo Investigator system uses a drawing tube to project the image of the counting frame onto the optical image. Thus the counting frame can be seen while looking through the microscope eyepieces. This system provides the highest resolution because the image degradation that occurs with video and digital cameras is eliminated. There are a variety of systems available, and each system is constantly being upgraded by the manufacturers. In general terms, factors to consider in choosing a system include (1) optical resolution, (2) accuracy of the counting system, (3) availability of service through the distributor, and (4) ease of use of the software (this is often a personal choice). A list of commercially available stereology programs is listed in Table 1.13.1. Using the dopaminergic projection from the SNpc to the CPu as a representative system, this unit provides methods for tissue preparation and histology (Basic Protocol 1), neuroanatomy (Basic Protocol 2), the stereological fractionator design (Basic Protocol 3), and a method for estimating an index of TT size in rats and mice of various genotypes (Basic Protocol 4). The main principles are to establish the neuroanatomy of the relevant nuclei and to establish a consistent counting technique. By way of example, the boundaries of the dorsal CPu (the target) and the SNpc (the source) are described. NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals. TT = T N Equation 1.13.1 Supplement 27 Contributed by David I. Finkelstein, Davor Stanic, Clare L. Parish, John Drago, and Malcolm K. Horne Current Protocols in Neuroscience (2004) 1.13.1-1.13.16 Copyright © 2004 by John Wiley & Sons, Inc. 1.13.1 Neuroanatomical Techniques